Nicotine-mediated signals modulate cell death and survival of T lymphocytes.

The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma.

BACKGROUND: The etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease. METHODS: We first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR. RESULTS: Each of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma). CONCLUSIONS: The data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.

Influence of genetic background on tumor karyotypes: evidence for breed-associated cytogenetic aberrations in canine appendicular osteosarcoma.

Recurrent chromosomal aberrations in solid tumors can reveal the genetic pathways involved in the evolution of a malignancy and in some cases predict biological behavior. However, the role of individual genetic backgrounds in shaping karyotypes of sporadic tumors is unknown. The genetic structure of purebred dog breeds, coupled with their susceptibility to spontaneous cancers, provides a robust model with which to address this question. We tested the hypothesis that there is an association between breed and the distribution of genomic copy number imbalances in naturally occurring canine tumors through assessment of a cohort of Golden Retrievers and Rottweilers diagnosed with spontaneous appendicular osteosarcoma. Our findings reveal significant correlations between breed and tumor karyotypes that are independent of gender, age at diagnosis, and histological classification. These data indicate for the first time that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes in a cancer with extensive genomic instability.

IL12 Abrogates Calcineurin-Dependent Immune Evasion during Leukemia Progression.

Exploitation of the immune system has emerged as an important therapeutic strategy for acute lymphoblastic leukemia (ALL). However, the mechanisms of immune evasion during leukemia progression remain poorly understood. We sought to understand the role of calcineurin in ALL and observed that depletion of calcineurin B (CnB) in leukemia cells dramatically prolongs survival in immune-competent but not immune-deficient recipients. Immune-competent recipients were protected from challenge with leukemia if they were first immunized with CnB-deficient leukemia, suggesting robust adaptive immunity. In the bone marrow (BM), recipients of CnB-deficient leukemia harbored expanded T-cell populations as compared with controls. Gene expression analyses of leukemia cells extracted from the BM identified Cn-dependent significant changes in the expression of immunoregulatory genes. Increased secretion of IL12 from CnB-deficient leukemia cells was sufficient to induce T-cell activation ex vivo, an effect that was abolished when IL12 was neutralized. Strikingly, recombinant IL12 prolonged survival of mice challenged with highly aggressive B-ALL. Moreover, gene expression analyses from children with ALL showed that patients with higher expression of either IL12A or IL12B exhibited prolonged survival. These data suggest that leukemia cells are dependent upon calcineurin for immune evasion by restricting the regulation of proinflammatory genes, particularly IL12. SIGNIFICANCE: This report implicates calcineurin as an intracellular signaling molecule responsible for immune evasion during leukemia progression and raises the prospect of re-examining IL12 as a therapeutic in leukemia.

Increased activity of both CDK1 and CDK2 is necessary for the combinatorial activity of WEE1 inhibition and cytarabine.

Inhibition of WEE1 is emerging as a promising chemosensitization strategy in many cancers including acute leukemia. Our lab and others have demonstrated that a small-molecule inhibitor of WEE1, AZD1775, sensitizes acute leukemia cells to cytarabine; however, a mechanism of combinatorial activity has remained elusive. Thus, we sought to determine the relative contribution of WEE1 targets CDK1 and CDK2 to the combinatorial activity of AZD1775 and cytarabine. To accomplish this, we expressed "WEE1 resistant" CDK1 (CDK1-AF) and CDK2 (CDK2-AF) constructs in a T-ALL cell line. Expression of CDK1/2-AF together, but neither alone, enhanced the anti-proliferative effects, DNA damage and apoptosis induced by cytarabine. Furthermore, pharmacologic inhibition of CDK1 alone or CDK1 and CDK2 together reduced the combinatorial activity of AZD1775 and cytarabine. Thus, increased activity of both CDK1 and CDK2 in response to WEE1 inhibition is necessary for the combinatorial activity of AZD1775 and cytarabine. This suggests the role of WEE1 in cells with accumulated DNA damage extends beyond regulation of CDK1 and the G2/M checkpoint and highlights the importance of WEE1 in mediating progression through the cell cycle.

Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance.

BACKGROUND: The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (approximately 90% in metastatic cases), and these tumors frequently contain PTEN abnormalities. RESULTS: Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. CONCLUSION: Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.

A Small-Molecule Inhibitor of WEE1, AZD1775, Synergizes with Olaparib by Impairing Homologous Recombination and Enhancing DNA Damage and Apoptosis in Acute Leukemia.

Although some patients with acute leukemia have good prognoses, the prognosis of adult and pediatric patients who relapse or cannot tolerate standard chemotherapy is poor. Inhibition of WEE1 with AZD1775 has been shown to sensitize cancer cells to genotoxic chemotherapies, including cytarabine in acute myeloid leukemia (AML) and T-ALL. Inhibition of WEE1 impairs homologous recombination by indirectly inhibiting BRCA2. Thus, we sought to determine whether AZD1775 could sensitize cells to the PARP1/2 inhibitor olaparib. We found that combined treatment with AZD1775 and olaparib was synergistic in AML and ALL cells, and this combination impaired proliferative capacity upon drug withdrawal. AZD1775 impaired homologous recombination in olaparib-treated cells, resulting in enhanced DNA damage accumulation and apoptosis induction. This combination enhanced disease control and increased survival in a murine AML model. Furthermore, we demonstrated that combined treatment with AZD1775 and olaparib reduces proliferation and colony formation and increases apoptosis in AML patient samples. In aggregate, these studies raise the possibility of rational combinations of targeted agents for leukemia in patients for whom conventional chemotherapeutics may not be effective or well tolerated. Mol Cancer Ther; 16(10); 2058-68. ©2017 AACR.

Enhancing antimelanoma immune responses through apoptosis.

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas(+) melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery.

STAT3-mediated autophagy dependence identifies subtypes of breast cancer where autophagy inhibition can be efficacious.

Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases, including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition, especially in combination with other therapy, could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for survival even in nutrient rich conditions without any additional stress, whereas others need autophagy only when stressed. Survival under unstressed conditions is due to cell type-specific autophagy regulation of STAT3 activity and this phenotype is enriched in triple-negative cell lines. This autophagy-dependency affects response to therapy because autophagy inhibition reduced tumor growth in vivo in autophagy-dependent but not in autophagy-independent breast tumors, whereas combination treatment with autophagy inhibitors and other agent was preferentially synergistic in autophagy-dependent cells. These results imply that autophagy-dependence represents a tumor cell-specific characteristic where autophagy inhibition will be more effective. Moreover, our results suggest that autophagy inhibition might be a potential therapeutic strategy for triple-negative breast cancers, which currently lack an effective targeted treatment.

Inhibition of Wee1 sensitizes cancer cells to antimetabolite chemotherapeutics in vitro and in vivo, independent of p53 functionality.

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia.

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.

Nicotine activates nuclear factor of activated T cells c2 (NFATc2) and prevents cell cycle entry in T cells.

We used primary peripheral blood T cells, a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously, to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly, T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that, in the face of chronic nicotine exposure, selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases.

Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium.

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (alpha(v)beta(3) integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.